The 3'??5' deoxyribonucleases are essential enzymes in DNA metabolism that catalyze excision of nucleotides from the 3' ends of DNA to prepare these 3' termini for subsequent steps during DNA processing. The long term goal of this project is to understand the biochemistry of the 3'??5' deoxyribonucleases encoded by the TREX genes that function in human cells and to elucidate the DNA metabolic pathways in which these enzymes function. The existence of 3' deoxyribonucleases in human cells has been recognized for almost forty years, but the biological functions of these enzymes are not well described. In the previous funding period we determined the structures of the TREX1 and TREX2 enzymes allowing us to begin to describe the biochemical properties of these 3' deoxyribonucleases. The TREX1 and TREX2 genes encode two structurally similar dimeric 3' deoxyribonucleases. The TREX1 enzyme contains a C-terminal region that is not found in TREX2, and the presence of two TREX genes is only found in mammals. Our biochemical studies to define the precise molecular characteristics of TREX1 have allowed us to quantify the activities of mutant TREX1 enzymes identified in patients diagnosed with the autoimmune diseases Aicardi- Goutieres syndrome, familial chilblain lupus, and in some cases of systemic lupus erythematosus. The finding of TREX1 mutations in autoimmune disease patients parallels the finding of TREX1 participation in apoptotic cell death. In this competitive renewal we will utilize the molecular tools we have developed in the previous funding period to focus our studies on the mechanisms of TREX1 action within cell death pathways. These studies will further our understanding of TREX1 exonuclease processing of DNA in cells by identifying the physiological substrate of TREX1 and will inform us of the mechanisms of TREX1 dysfunction that cause the pathological findings associated with autoimmune disease. Our efforts to understand the mechanisms of TREX1 biochemistry and cellular function are addressed according to the following aims. In Aim 1 we will determine the mechanism of TREX1 action within the catalytic core. In Aim 2, we will determine the effects of TREX1 on chromatin disassembly in cell death pathways. In Aim 3 we will determine the mechanism of TREX1 targeting to the ER. PUBLIC HEALTH REVELANCE: This grant proposal is to study how enzymes process DNA and to determine how dysfunction of these enzymes causes autoimmune disease. The outcome of the proposed research will have a significant impact on the medical treatment of complex autoimmune diseases such as systemic lupus erythematosus.